The proteins initiated in vitro by nascent bacteriophage f2 RNA strands attached to isolated replicating structures have been analyzed. The observations confirm that coat protein is the major product initiated and completed. Nascent strands direct the initiation of viral maturation protein in amounts similar to the maximum levels observed in vivo; this synthesis is independent of translation of the coat protein gene. However, only a fraction of these maturation protein molecules initiated in vitro is completed. Nascent RNA molecules also direct the initiation of appreciable amounts of viral RNA polymerase protein, very little of which is completed. Certain constraints on the in vitro translation of the polymerase gene from single-stranded RNA appear to be relaxed in the nascent strands, as indicated by the reduced effect of a polar amber mutation in the coat cistron upon polymerase protein initiation from nascent RNA.